Development and application of a scalable workflow for immunomagnetic separation of exRNA carrier subclasses and molecular analysis of their cargo

总结 细胞外RNA（exRNA）已被发现在所有测试的人类生物流体，有越来越多的证据表明， 它们可以作为细胞间通讯的介质，以及诊断，预后， 用于广泛疾病和生理状况的治疗诊断生物标志物。ExRNA与 多种载体亚类（CS），包括细胞外囊泡（EV），核糖核蛋白复合物（RNP）， 和脂蛋白（LPP），其中许多是未知的或表征不佳的。这个跨学科的团队 在exRNA和脂蛋白生物学、exRNA生物标志物发现、exRNA治疗、exRNA 测序，低输入蛋白质组学和脂质组学，以及流式细胞术，将共同努力，开发和应用一个 严格、可重复、高效、可扩展且具有成本效益的免疫磁性分离（IMS）工作流程， 制备性分离CS用于下游组学分析。此外，基于多重珠粒的 将探索用于更有效地分离CS的流动分选。目标1A将侧重于发展 使用适当的细胞培养模型鉴定和分离已知和疑似CS的试剂 和健康人血浆和血清样品。这项工作将包括筛选可用的抗体， 已知的一般CS（例如四跨膜蛋白、AGO蛋白、载脂蛋白）和多种细胞类型的标记物- 特异性标记物，用于识别在Western Blot和IMS中表现良好的抗体，并传播结果 成功和失败的抗体。在目标1B中，目标1A的结果将用于建立和 测试IMS工作流程，用于分离和小分子和长分子RNAseq、蛋白质组学和脂质组学分析， CS和小区类型特定EV。除了建立一个全面的知识集涵盖RNA， 已知和疑似CS的蛋白质和脂质货物，分析IMS未捕获的物质 工作流程将揭示新的CS。目标2A将包括完善国际监测系统的工作流程， 临床队列：妊娠和非妊娠女性对照;心肌梗死后和年龄-和性别- 匹配的风险对照;以及上皮性卵巢癌和年龄匹配的健康女性对照。分析 通过小和长RNAseq与一般CS和细胞类型特异性EV相关的exRNA将揭示 是否这些CS的相对丰度和exRNA货物在这些病例和对照之间存在差异， 同伙目标2B将包括开发一种基于流式细胞术的多重同时检测策略。 从人血浆和血清中分离多种CS。如果成功，该项目将导致发展 用于分离exRNA CS的严格工作流程，可重复且快速地产生 在临床上以具有成本效益的方式高度富集所需的CS，同时最大限度地减少其他CS的污染 输入材料的可行体积，并产生足够的材料用于下游分子分析。此外，本发明还提供了一种方法， 在该项目实施过程中产生的全面的omic数据将产生有价值的参考概况 先前已知的和新的CS携带的RNA、蛋白质和脂质货物的数据。

SUMMARY Extracellular RNAs (exRNAs) have been found in all tested human biofluids, and there is increasing evidence that they can serve as mediators of intercellular communication, as well as diagnostic, prognostic, and theranostic biomarkers for a wide range of disease and physiological conditions. ExRNAs are associated with a variety of carriers subclasses (CSs), including extracellular vesicles (EVs), ribonucleoprotein complexes (RNPs), and lipoproteins (LPP), many of which are as-yet unknown or poorly characterized. This transdisciplinary team, with expertise in exRNA and lipoprotein biology, exRNA biomarker discovery, exRNA therapeutics, exRNA sequencing, low-input proteomics and lipidomics, and flow cytometry, will work together to develop and apply a rigorous, reproducible, efficient, scalable, and cost-effective immunomagnetic separation (IMS) workflow for preparative isolation of CSs for downstream omic analysis. In addition, the potential for multiplex bead-based flow sorting for even more efficient separation of CSs will be explored. Aim 1A will focus on development of reagents for identification and separation of known and suspected CSs using appropriate cell culture models and healthy human plasma and serum samples. This work will include screening of available antibodies against markers for known general CSs (e.g. tetraspanins, AGO proteins, apolipoproteins) and a variety of cell type- specific markers to identify antibodies that perform well for Western Blot and IMS, and dissemination of results for both successful and unsuccessful antibodies. In Aim 1B, the results from Aim 1A will be applied to build and test an IMS workflow for separation and small and long RNAseq, proteomic, and lipidomic analysis of general CSs and cell type-specific EVs. In addition to building a comprehensive knowledge set encompassing the RNA, protein, and lipid cargo of known and suspected CSs, profiling the material that is not captured by the IMS workflow will reveal novel CSs. Aim 2A will encompass refinement of the IMS workflow and application to three clinical cohorts: Pregnant and non-pregnant female controls; Post-myocardial infarction and age- and sex- matched at-risk controls; and Epithelial ovarian cancer and age-matched healthy female controls. Analysis of the exRNAs associated with general CSs and cell type-specific EVs by small and long RNAseq will reveal whether the relative abundance and exRNA cargo of of these CSs differs between cases and controls in these cohorts. Aim 2B will consist of development of a flow cytometry-based strategy for multiplexed simultaneous separation of multiple CSs from human plasma and serum. If successful, this project will result in development of a rigorous workflow for separation of exRNA CSs that reproducibly and rapidly produces fractions that are highly enriched for desired CSs with minimal contamination by other CSs in a cost-effective manner on clinically feasible volumes of input material, and yields sufficient material for downstream molecular analysis. In addition, the comprehensive omic data generated on during the course of this project will yield valuable reference profiling data on the RNA, protein, and lipid cargo carried by previously known and novel CSs.